Ethanol activates several classes of G-proteins leading to the stimulation of several intracellular signal transduction pathways, including adenylyl cyclase, phospholipase C and ion channels. On a chronic basis, ethanol induces profound alterations in the expression of diverse G-proteins with associated functional consequences. The mechanisms by which ethanol affects G-protein activity and expression is only partially understood. Recent studies have demonstrated that ethanol markedly interferes with dynamic G-protein palmitoylation, which is important for plasma membrane localization and modulates the interaction of alpha subunits with effectors. The overall hypothesis of this proposal is that the derangement of the G-protein palmitoylation cycle by ethanol contributes to its effects on G-protein activity and expression. These issues will be investigated in neuronal NG-108 cells and cultured rat cerebellar granule cells under basal and receptor-stimulated conditions. Palmitoylation of the alpha subunits that are functionally affected by ethanol will be studied (Gs, Gi and Gq). Possible sites of ethanol inhibition to be studied included palmitoyl acylthioesterase (depalmitoylation) and palmitoyl acyltransferase (palmitoylation). The effect of ethanol on palmitoyl alpha subunit pool sizes will be determined by HPLC. The functional consequences of the acute ethanol effect will be assessed by analyzing the distribution of alpha subunits in the cytosol and in the plasma membranes. A mutational cDNA approach will be used to assess the role of G-protein palmitoylation on the stimulation of effectors using intact cells and plasma membrane preparations. We will explore the hypothesis that chronic ethanol treatment reduces the specific pool of palmitoylated G-protein available for effector stimulation, including a specific pool that appears to be associated with tyrosine kinase receptors. The pool sizes of alpha subunits will be measured in membranes and cytosol from neural cells treated chronically with ethanol in vitro, and in cells obtained from rats fed ethanol chronically. Finally, the effect of chronic ethanol treatment on dynamic palmitoylation will be assessed for various G-protein classes. In summary, the proposed studies are designed to relate ethanol-induced structural alterations in a family of key signal transduction molecules leading to diverse functional effects on acute and chronic bases.